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Sample GSM6938265 Query DataSets for GSM6938265
Status Public on Jan 20, 2023
Title ETS1_UV
Sample type protein
Source name Human ETS1
Organism Homo sapiens
Characteristics protein: ETS1
protein concentration: 100nM
array condition: UV
Growth protocol Cell growth protocol is detailed in the manuscript for all tested proteins and antibodies.
Extracted molecule protein
Extraction protocol Extract protocol is detailed in the manuscript for all tested proteins and antibodies.
Label Alexa 635
Label protocol Label protocol is detailed in the manuscript for all tested proteins and antibodies.
Hybridization protocol Commercially synthesized, high-density, single-stranded DNA microarrays from Agilent were first double-stranded via primer extension as done in previous assays (Berger et al, Nature Biotechnology 2006; Shen et al, Cell Systems 2018). The arrays were partially irradiated with UVC such that only some chambers were exposed to UVC while others were not. In order to irradiate only certain parts of the array, and protect the rest from UV irradiation, a cover/gasket slide was cut to the desired size are used to partially cover the DNA array. Next, the gasket slide was fully covered using Cryo-Babies® labels, and the array-gasket slide sandwich was placed in an open container with 1x PBS. To irradiate the DNA on the array, we used a Stratalinker® UV Crosslinker 1800 instrument and conducted a series of irradiations with UVC. Using the energy mode, the container was irradiated with 100 J/m2, randomly repositioned inside the crosslinker 15 times to achieve 1,500 J/m2. Midway through the series, the PBS was replaced in order to keep the solution at room temperature. This procedure resulted in a DNA array with some chambers of the array irradiated by UV and others not irradiated. Samples with the “UV” condition used irradiated chambers while those with a “Non-UV” condition used non-irradiated chambers. The arrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk for 1 hour as done in prior assays (Shen et al, Cell Systems 2018). Proteins were incubated at the indicated concentration using the buffers referred to in the manuscript for 1 hour. Microarrays were again washed once with PBS / 0.5% (vol/vol) Tween-20 for 3 min, and then once with PBS / 0.01% Triton X-100 for 2 min (Shen et al, Cell Systems 2018). Then the arrays were incubated with either Penta-His Alexa488-conjugated antibody and Penta-His Alexa647-conjugated antibody (1:20 and 1:40 ratio respectively) in a buffer with 2% (wt/vol) milk and 0.05% Tween for 1 hour (buffer details described in the manuscript). After incubation, the arrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min as done in prior work (Shen et al, Cell Systems 2018).
Scan protocol Protein bound microarrays were scanned at 635nm using a GenePix 4400A at multiple laser power and gain settings to best capture the signal and avoid saturation of spots. The resulting TIF images were processed using GenePix Pro 7.3.
Data processing The raw data collected using the GenePix® 4400A microarray scanner was processed using the Seed and Wobble suite (Berger et al, Nature Biotechnology 2006), with modifications to adapt the k-mer data to our UV-Bind universal design, as described in the manuscript. For each unique sequence tested, median values over the spots containing that sequence were used unless otherwise noted (i.e. for the universal design sequences and for the competition tests). The number of replicate spots for each sequence varied between 3 and 20, depending on the DNA library. Processing of k-mers and PWMs using Seed-and-Wobble was done using the adjusted signal (i.e. Alexa488Adjusted or Alexa635Adjusted column) in the alldata.txt file as done by default (Berger et al, Nature Biotechnology 2006).
Submission date Jan 17, 2023
Last update date Jan 20, 2023
Contact name Raluca Gordan
Organization name Duke University
Department Center for Genomic and Computational Biology
Street address 101 Science Dr, CIEMAS 2179
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
Platform ID GPL33026
Series (2)
GSE223013 UV irradiation remodels the specificity landscape of transcription factors [84702_Proteins]
GSE223023 UV irradiation remodels the specificity landscape of transcription factors

Data table header descriptions
VALUE Signal intensity

Data table
Ctrl_UUV_000001 4752
Ctrl_UUV_000002 14378
Ctrl_UUV_000003 7677
Ctrl_UUV_000004 7258
Ctrl_UUV_000005 12140
Ctrl_UUV_000006 13062
Ctrl_UUV_000007 14200
Ctrl_UUV_000008 5397
Ctrl_UUV_000009 8656
Ctrl_UUV_000010 11779
Ctrl_UUV_000011 11685
Ctrl_UUV_000012 5253
Ctrl_UUV_000013 8783
Ctrl_UUV_000014 8795
Ctrl_UUV_000015 10907
Ctrl_UUV_000016 10593
Ctrl_UUV_000017 15452
Ctrl_UUV_000018 12654
Ctrl_UUV_000019 4152
Ctrl_UUV_000020 14151

Total number of rows: 175866

Table truncated, full table size 3623 Kbytes.

Supplementary file Size Download File type/resource
GSM6938265_ETS1_UV_84702_alldata.txt.gz 6.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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